Oligomers In Solution Research Articles

Heat-induced fibrillation of BclXL apoptotic repressor

The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease.

Analysis of the Oligomeric State of the Mating ATPase ConE and its Arginine Finger Mutant

During conjugation, mating pores allow bacteria to transfer DNA to recipient cells. ConE is one of several proteins that make up the mating pore of Bacillus subtilis. ConE, and its‐characterized homologs VirB4 and TrwK, belongs to the FtsK‐HerA superfamily of ATPases which contain a conserved arginine finger that is a good predictor for ring formation and inter‐protomer interactions. Several superfamily members have been purified and characterized in vitro; they form monomers, dimers, and higher order oligomers in solution and generally form hexamers when crystallized. We tested if ConE oligomerizes and how the mutation of ConE's arginine finger affects its oligomerization. His6‐ConE and mutant His6‐ConE (R726A) were purified via nickel affinity chromatography. Purification was monitored via SDS‐PAGE and multimeric states were determined by BN‐PAGE. We found that both wild type and mutant His6‐ConE form monomers, dimers, and higher oligomers. Addition of ATP, single‐stranded DNA, and double‐stranded DNA did not dramatically alter the oligomeric states.

Investigation of protein oligomerization and assembly of a DNA translocation channel

Mating, or conjugation, is the process by which a cell horizontally passes genetic information to another cell through a multi‐protein DNA translocation channel. ConE is a key ATPase of the mating machinery of ICEBs1, the Integrative and Conjugative element of Bacillus subtilis. Characterized homologs form a variety of oligomers in solution and crystallize as hexamers. Previously, we showed that purified His6‐ConE is a mixture of dimers, trimers, and higher‐order oligomers via Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE). To more thoroughly understand the interactions between ConE and other players in the mating machinery, we have employed a bacterial two‐hybrid system. We investigated the interaction between ConE fused to the T18 and T25 domains of Bordetella pertussis adenylate cyclase. Using β‐galactosidase assay to indirectly report cyclic‐AMP levels, we found that T25‐ConE and T18‐ConE interact. We hope to use this technique to measure possible interactions between ConE and other ICEBs1 proteins that make up the DNA translocation channel. This research was funded by an NSF‐RUI grant to Melanie B. Berkmen.

Propagation of Chirality in Mixtures of Natural and Enantiomeric DNA Oligomers

Concentrated solutions of ultrashort duplex-forming DNA oligomers may develop various forms of liquid crystal ordering among which is the chiral nematic phase, characterized by a macroscopic helical precession of molecular orientation. The specifics of how chirality propagates from the molecular to the mesoscale is still unclear, both in general and in the case of DNA-based liquid crystals. We have here investigated the onset of nematic ordering and its chiral character in mixtures of natural D-DNA oligomers forming right-handed duplex helices and of mirror symmetric (L-DNA) molecules, forming left-handed helices. Since the nematic ordering of DNA duplexes is mediated by their end-to-end aggregation into linear columns, by controlling the terminals of both enantiomers we could study the propagation of chirality in solutions where the D and L species form mixtures of homochiral columns, and in solutions of heterochiral columns. The two systems behave in markedly different fashion. By adopting a simple model based on nearest-neighbor interactions, we account for the different observed dependence of the chirality of these two systems on the enantiomeric ratio.

Consolidation of calcareous and siliceous sandstones by hydroxyapatite: Comparison with a TEOS-based consolidant

Abstract A new consolidating treatment for limestone, based on the reaction between diammonium hydrogen phosphate (DAP) and calcite in the stone to form hydroxyapatite (HAP), has recently been proposed. Thanks to the formation of HAP at grain boundaries, so that grains are more effectively bonded, HAP-treated limestone undergoes a significant improvement in mechanical properties. Considering that the HAP treatment has the advantage of not substantially altering the transport properties of treated stones, unlike many other consolidating products currently used, in this paper, the effectiveness of HAP as a consolidant for sandstones with varying carbonate content and porosity was investigated and compared with that of a TEOS-based consolidant. Two lithotypes were used: Giallo Siena (a highly porous calcareous sandstone) and Pietra Serena (a quartzitic sandstone with low porosity). After preliminary artificial weathering according to previously developed methodologies, each lithotype was treated: (i) with a 1 M aqueous solution of DAP; (ii) with a solution of TEOS oligomers in isopropyl alcohol, each applied by brushing until apparent refusal. The effects of the two consolidating treatments were assessed by comparing microstructural, physical and mechanical properties of treated and untreated stones. HAP-treated specimens experienced an increase in dynamic elastic modulus and tensile strength substantially similar to that achieved by TEOS-treated ones. However, differently from samples consolidated with TEOS, HAP-treated stones experienced very low variations in porosity and pore size distribution, leading to substantially no variation in the rate of water sorption and only a small decrease in water vapor permeability. Considering the good performance on both lithotypes, comparable to that achieved by a TEOS-based treatment, HAP is confirmed as a promising consolidating treatment, which has the advantage of being effective after just two days, not substantially altering the transport properties of treated stones and not being hazardous for human health, thanks to its aqueous solvent.

A sequence-dependent rigid-base model of DNA

A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can successfully predict the nonlocal changes in the minimum energy configuration of an oligomer that are consequent upon a local change of sequence at the level of a single point mutation.

Antioxidant activity of Maillard reaction products derived from aqueous and ethanolic glucose-glycine and its oligomer solutions

The purpose of this study was to evaluate the antioxidant activity of Maillard reaction products (MRPs) in both aqueous and ethanolic glucose-glycine oligomer solutions. The reduction in pH was higher in aqueous MRP solutions than ethanolic MRP solutions. The samples in ethanolic MRP solutions had greater Abs294 nm and Abs420 nm than those in aqueous MRP solutions. The ferrous ion chelating activity of all MRP samples was much higher than the cupric ion chelating ability in both aqueous and ethanolic solutions. The antioxidant activity in ethanolic MRP solutions was higher than that in aqueous MRP solutions. MRPs derived from the diglycine were found to be effective antioxidants in different in vitro assays with regard to the ABTS and DPPH radical scavenging activities, and ferric reducing/antioxidant power.

Effect of the structure of a network matrix on the formation of gold nanoparticles during photoreduction of its salts in solutions of acrylic oligomers

The effect of network parameters of three-dimensionally crosslinked polyacrylic optically transparent matrices on the generation of surface plasmon resonance of gold nanoparticles forming during irradiation of Au3+ salt solutions in a liquid acrylic composite is studied. During the UV irradiation of a solution, the photoreduction of gold and the photoinitiated polymerization of acrylates proceed simultaneously to form transparent colorless films in which, after subsequent light and/or heat treatment, an absorption band near 550 nm that corresponds to the plasmon resonance of nanosize metal particles is generated. During the photoreduction of gold salt in matrices of oligo(urethane acrylate) composites with similar structures of the oligomeric block, the parameters of the three-dimensional network—the internodal and interchain distances—affect the rate of generation of gold nanoparticles but not the limiting value of optical density for the plasmon-resonance band of nanoparticles. Polyacrylate matrices may serve as a registering medium for optical data recording.

Synaptotoxic Amyloid-β Oligomers: A Molecular Basis for the Cause, Diagnosis, and Treatment of Alzheimer's Disease?

The oligomer hypothesis for Alzheimer's disease (AD)was introduced in 1998. It was based on evidence that oligomers could exist free of amyloid fibrils, that fibril-free oligomer solutions rapidly inhibited long term potentiation, and that oligomers ultimately caused a highly selective nerve cell death. Fibrils no longer were the only toxins made by amyloid-β (Aβ), and likely not the most important ones. Oligomers provided a new basis for instigating AD. Since introduction of the hypothesis, more than 1,500 articles on oligomers have been published. Articles for this review were selected for contributions to oligomer theory at three different levels. The first set demonstrated new aspects of oligomer pathobiology in cell models, showing that exposure of neurons to oligomers is sufficient to cause key features of AD neuropathology. The second set confirmed the relationship between oligomers and salient AD neuropathology in animal models, consistent with other in vivo studies that overall have substantiated cell-based discoveries. The third set developed strategies for therapeutic targeting of oligomers, introducing both small molecule and antibody-based approaches. These and related findings from many groups have helped establish oligomers as central to the mechanism of AD pathogenesis. Comprising a ligand-based attack on specific synapses, the action of toxic oligomers gives a molecular basis to account for key features of AD neuropathology and to explain why early disease targets memory. Although there still is no effective treatment for AD, insights over the past five years raise hopes that new approaches targeting Aβ oligomers could finally bring disease-modifying therapeutics.

Modulation of CD147-induced matrix metalloproteinase activity: role of CD147 N-glycosylation

Degradation of the basement membrane by MMPs (matrix metalloproteinases) is one of the most critical steps in tumour progression. CD147 is a tumour-associated antigen that plays a key regulatory role for MMP activities. In the present study, mass spectrum analysis demonstrated that the purified native CD147 from human lung cancer tissue was N-glycosylated and contained a series of high-mannose and complex-type N-linked glycan structures. Moreover, native glycosylated CD147 existed exclusively as oligomers in solution and directly stimulated MMP production more efficiently than non-glycosylated prokaryotic CD147. The glycosylation site mutation results indicated that, among three N-glycan attachment sites, the N152Q mutants were retained in the endoplasmic reticulum and unfolded protein response signalling was activated. This improper intracellular accumulation impaired its MMP-inducing activity. Increased β1,6-branching of N-glycans as a result of overexpression of GnT-V (N-acetylglucosaminyltransferase V) plays an important role in tumour metastasis. In the present study, we identified CD147 as a target protein of GnT-V and found that overexpression of GnT-V resulted in an elevated level of CD147 at the plasma membrane and in cell-conditioned medium, thereby increasing the induction of MMPs. The present study reveals the important role of N-glycosylation of CD147 in its biological function and implied that targeting aberrant β1,6-branching of N-glycans on CD147 would be valuable for the development of novel therapeutic modalities against carcinoma.

The major mRNP protein YB-1: Structural and association properties in solution

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional 1H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.

Crystal structure of the dimeric coiled-coil domain of the cytosolic nucleic acid sensor LRRFIP1

LRRFIP1 binds cytoplasmic double-stranded DNA and RNA and interacts with FLI, the mammalian homolog of Drosophila flightless I, through a highly conserved 87-amino acid domain. Upon binding nucleic acid ligands, LRRFIP1 recruits and activates β-catenin, leading to the IRF3-dependent production of type I interferon. However, the molecular mechanism of LRRFIP1 signaling is not well understood. Here we show that the FLI-interacting domain of LRRFIP1 forms a classic parallel, homodimeric coiled coil with 10 heptad repeats and 22 helical turns. The coiled coil domain is also a dimer in solution. However, a longer LRRFIP1 construct spanning the coiled coil and DNA binding domains assembles into higher order oligomers in solution. The structure of LRRFIP1-CC constitutes a valuable tool for probing the mechanism of LRRFIP1 signaling and for structural studies of larger LRRFIP1 constructs.

Self-Assembly, Chromic Properties, and Nanostructure Formation of Tetrathiafulvalene-Fused Dodecadehydro[18]annulenes

Abstract Multifunctional dodecadehydrotris(tetrathiafulvaleno)[18]annulenes 1b–1d, which have six peripheral butylthio substituents or six ester substituents, were synthesized together with the corresponding [12]annulene 2c. The [18]annulenes 1b–1d easily formed stacked dimers and higher oligomers in solution via nanoscopic phase separation among the π-frame, ester or alkylthio substituents, and inner sphere to show solvatochromism and thermochromism. Furthermore, the strong self-aggregation of their oxidized radical cations in solution resulted in electrochromism depending on the formation of various types of mixed-valence states and π-dimers. Interestingly, self-aggregation of the [18]annulenes 1b–1d in amphiphilic media such as THF–H2O either produced fibrous structures in the case of the hexabutylthio derivative 1b and hexabutyl ester 1c or resulted in the temperature hysteresis of the color, UV–vis absorptions, and 1H NMR signals obtained in solution in the case of the hexaoctyl ester 1d which forms no fiber and precipitate under similar conditions. It is noted that fibers of the hexabutyl ester 1c can be aligned in a magnetic field using the large diamagnetic response of the [18]annulene frame for the driving force. A small difference in the substituents of 1b–1d causes a remarkable change in either their solvato- and electrochromism or nanostructure formation of 1b–1d based on the self-assembling nature both in solution and in the solid state.

Highly soluble phenylethynyl-terminated imide oligomers based on KAPTON-type backbone structures for carbon fiber-reinforced composites with high heat resistance

Novel addition-type phenylethynyl-containing imide oligomers (degree of polymerization: n=1–10) derived from 1,2,4,5-benzenetetracarboxylic dianhydride or pyromellitic dianhydride (PMDA), 2-phenyl-(4,4′-diaminodiphenyl ether) (p-ODA) and 4-phenylethynylphthalic anhydride (PEPA) were prepared and evaluated as a matrix resin for high heat-resistant carbon fiber-reinforced composites. The uncured imide oligomers showed good solubility (>30 wt%) in N-methyl-2-pyrrolidone and very low melt viscosities. These imide oligomers were successfully converted to crosslinked structures after curing at 370 °C. The glass transition temperature (Tg) and the elongation at break (ɛb) of the cured imide resin were found to be excellent (almost 340 °C and >15%, respectively). The carbon fiber prepreg was prepared from the imide oligomer solution and used to fabricate void-free laminates with high Tg. The thermal and mechanical properties of the laminates were determined at ambient temperature.

Preparation and characterization of monolithic methylsilicone xerogels doped with liquid-phase synthesized TiO2

The xerogels of methylsilicone doped with TiO2 were prepared by incorporation of liquid-phase prepared TiO2 in methylsilicone matrix. The microstructure and properties of the composite xerogels were investigated by Fourier transform infrared spectra (FTIR), scanning electron microscopy (SEM), N2 sorption, and wide-angle X-ray powder diffraction (XRD). The results indicated that the as-prepared TiO2 particles could be well dispersed in methylsilicone matrix. The different amounts of TiO2 doped in methylsilicone oligomer solution resulted in different reaction process and microstructure. The thermal stability of the composite monolith was enhanced after doping TiO2 particles.

Cooperative and Noncooperative Assembly of Oligopyrenotides Resolved by Atomic Force Microscopy

The supramolecular assembly of amphiphilic oligopyrenotide building blocks (covalently linked heptapyrene, Py7) is studied by atomic force microscopy (AFM) in combination with optical spectroscopy. The assembly process is triggered in a controlled manner by increasing the ionic strength of the aqueous oligomer solution. Cooperative noncovalent interactions between individual oligomeric units lead to the formation of DNA-like supramolecular polymers. We also show that the terminal attachment of a single cytidine nucleotide to the heptapyrenotide (Py7-C) changes the association process from a cooperative (nucleation–elongation) to a noncooperative (isodesmic) regime, suggesting a structure misfit between the cytidine and the pyrene units. We also demonstrate that AFM enables the identification and characterization of minute concentrations of the supramolecular products, which was not accessible by conventional optical spectroscopy.

Synthesis of Spin‐Crossover Nano‐ and Micro‐objects in Homogeneous Media

New methods are proposed for the synthesis of spin-crossover nano- and micro-objects. Several nano-objects that are based upon the spin-crossover complex [Fe(hptrz)(3)](OTs)(2) (hptrz=4-heptyl-1,2,4-triazole, Ts=para-toluenesulfonyl) were prepared in homogeneous media. The use of various reagents (Triton X-100, PVP, TOPO, and PEGs of different molecular weights) as stabilizing agents yielded materials of different size (6 nm-2 μm) and morphology (nanorods, nanoplates, small spherical particles, and nano- and micro-crystals). In particular, when Triton X-100 was used, a variation in the morphology from nanorods to nanoplates was observed by changing the nature of the solvent. Interestingly, the preparation of the nanorods and nanoplates was always accompanied by the formation of small spherical particles. Alternatively, when PEG was used, 200-400 nm crystals of the complex were obtained. In addition, a very promising polymer-free synthetic method is discussed that was based on the preparation of relatively stable Fe(II)-triazole oligomers in CHCl(3). Their specific treatment led to micro-crystals, small nanoparticles, or gels. The size and morphology of all of these objects were characterized by TEM and by dynamic light scattering (DLS) where possible. Their spin-crossover behavior was studied by optical and magnetic measurements. The spin-transition features for large particles (>100 nm) were very similar to that of the bulk material, that is, close to room temperature with a hysteresis width of up to 8 K. The effects of the matrix and/or size-reduction led to modification of the transition temperature and an abruptness of the spin transition for oligomeric solutions and small nanoparticles of 6 nm in size.

Pyridine-cored V-shaped π-conjugated oligomers: synthesis and optical properties

A new series of V-shaped pyridine-cored π-conjugated oligomers are synthesized utilizing two-fold Heck/Suzuki coupling reactions. Optical properties of these compounds (λmax=390–449 nm, Φfl=79–5%, in solutions) are discussed. They are shown to be thermally stable and soluble in common organic solvents. Stilbenoid oligomers exhibited much higher fluorescence quantum yields than tri- and tetra-phenylethylene substituted oligomers in solutions.

Single-Particle Characterization of Aβ Oligomers in Solution

Determining the pathological role of amyloids in amyloid-associated diseases will require a method for characterizing the dynamic distributions in size and shape of amyloid oligomers with high resolution. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloid-β oligomers directly in solution and without chemical modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β aggregate sizes. The approach revealed the distribution of protofibrillar lengths (12- to 155 -mer) as well as the average cross-sectional area of protofibrils and fibers.

A conserved domain in the N‐terminus is important for LEAFY dimerization and function in Arabidopsis thaliana

The floral meristem identity gene LEAFY (LFY) of Arabidopsis thaliana is essential for the formation of fertile flowers and has roles in the control of several aspects of floral development, which include phyllotaxy and organ number and identity. This gene encodes a land plant-specific transcription factor and regulates expression of a number of genes that include other floral meristem identity genes and floral homeotic genes. Although the LFY DNA-binding domain has a structure that resembles that of helix-turn-helix proteins, LFY and its orthologs represent a novel family of transcription factors that are characterized by a conserved N-terminus domain of unknown function and a C-terminus DNA-binding domain. Many transcription factors act as dimers. These dimers are essential for the biological activity of the proteins. We demonstrate that LFY forms homodimers or oligomers in solution. This association is mediated through the N-terminus conserved region of the LFY protein. Although mutant LFY proteins that cannot dimerize in solution can bind DNA, the binding is weaker than that of wild type LFY protein. LFY-LFY interactions mediated by the N-terminus domain are essential for the biological activity of this protein, as mutations that abolish the ability to self-associate cannot complement an lfy null allele. Our data indicate: (i) that LFY, and probably its orthologs in other plants, must act in complexes that contain at least two LFY molecules; and (ii) that the N-terminus is essential for stabilization of LFY complexes. This situation is integral to the ability of LFY to regulate gene expression.